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Functions of Gle1 are governed by two distinct modes of self-association

2020; Elsevier BV; Volume: 295; Issue: 49 Linguagem: Inglês

10.1074/jbc.ra120.015715

1083-351X

Aaron C. Mason, Susan R. Wente,

Viral Infections and Immunology Research

Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis. Gle1 is a conserved, essential regulator of DEAD-box RNA helicases, with critical roles defined in mRNA export, translation initiation, translation termination, and stress granule formation. Mechanisms that specify which, where, and when DDXs are targeted by Gle1 are critical to understand. In addition to roles for stress-induced phosphorylation and inositol hexakisphosphate binding in specifying Gle1 function, Gle1 oligomerizes via its N-terminal domain in a phosphorylation-dependent manner. However, a thorough analysis of the role for Gle1 self-association is lacking. Here, we find that Gle1 self-association is driven by two distinct regions: a coiled-coil domain and a novel 10-amino acid aggregation-prone region, both of which are necessary for proper Gle1 oligomerization. By exogenous expression in HeLa cells, we tested the function of a series of mutations that impact the oligomerization domains of the Gle1A and Gle1B isoforms. Gle1 oligomerization is necessary for many, but not all aspects of Gle1A and Gle1B function, and the requirements for each interaction domain differ. Whereas the coiled-coil domain and aggregation-prone region additively contribute to competent mRNA export and stress granule formation, both self-association domains are independently required for regulation of translation under cellular stress. In contrast, Gle1 self-association is dispensable for phosphorylation and nonstressed translation initiation. Collectively, we reveal self-association functions as an additional mode of Gle1 regulation to ensure proper mRNA export and translation. This work also provides further insight into the mechanisms underlying human gle1 disease mutants found in prenatally lethal forms of arthrogryposis. Throughout the gene expression pathway, the fate of a protein-coding mRNA transcript is determined by interactions between its specific complement of RNA-binding proteins and other cellular machinery. During this process, stepwise changes to messenger ribonucleoprotein (mRNP) complexes are achieved through the remodeling actions of RNA-dependent DEAD-box ATPases (termed Dbp in Saccharomyces cerevisiae and DDX in humans) (1Xie Y. Ren Y. Mechanisms of nuclear mRNA export: a structural perspective.Traffic. 2019; 20 (31513326): 829-84010.1111/tra.12691Crossref PubMed Scopus (24) Google Scholar). Providing an additional layer of regulation, some Dbps/DDXs require specific cofactors that modulate their ATPase activities (2Sloan K.E. Bohnsack M.T. Unravelling the mechanisms of RNA helicase regulation.Trends Biochem. Sci. 2018; 43 (29486979): 237-25010.1016/j.tibs.2018.02.001Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Among the known cofactors, the essential conserved multidomain protein Gle1 is a uniquely versatile Dbp/DDX modulator. Gle1 is involved in facilitating proper nuclear mRNA export, translation, and the stress response (3Aditi Folkmann A.W. Wente S.R. Cytoplasmic hGle1A regulates stress granules by modulation of translation.Mol. Biol. Cell. 2015; 26 (25694449): 1476-149010.1091/mbc.E14-11-1523Crossref PubMed Scopus (25) Google Scholar, 4Alcázar-Román A.R. Tran E.J. Guo S. Wente S.R. Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.Nat. Cell Biol. 2006; 8 (16783363): 711-71610.1038/ncb1427Crossref PubMed Scopus (207) Google Scholar, 5Bolger T.A. Wente S.R. Gle1 is a multifunctional DEAD-box protein regulator that modulates Ded1 in translation initiation.J. Biol. Chem. 2011; 286 (21949122): 39750-3975910.1074/jbc.M111.299321Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 6Bolger T.A. Folkmann A.W. Tran E.J. Wente S.R. The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation.Cell. 2008; 134 (18724935): 624-63310.1016/j.cell.2008.06.027Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 8Jao L.-E. Akef A. Wente S.R. A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies.Mol. Biol. Cell. 2017; 28 (28035044): 120-12710.1091/mbc.E16-09-0675Crossref PubMed Google Scholar, 9Montpetit B. Thomsen N.D. Helmke K.J. Seeliger M.A. Berger J.M. Weis K. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP 6 in mRNA export.Nature. 2011; 472 (21441902): 238-24210.1038/nature09862Crossref PubMed Scopus (161) Google Scholar, 10Weirich C.S. Erzberger J.P. Flick J.S. Berger J.M. Thorner J. Weis K. Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.Nat. Cell Biol. 2006; 8 (16783364): 668-67610.1038/ncb1424Crossref PubMed Scopus (194) Google Scholar). In human cells, at least two different Gle1 isoforms, A and B, result from alternative splicing of a single GLE1 pre-mRNA transcript (11Kendirgi F. Barry D.M. Griffis E.R. Powers M.A. Wente S.R. An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.J. Cell Biol. 2003; 160 (12668658): 1029-104010.1083/jcb.200211081Crossref PubMed Scopus (46) Google Scholar). Differing exclusively in their C-terminal regions, only Gle1B contains a 39-amino acid extension that interacts with Nup42 on the cytoplasmic face of the nuclear pore complex (NPC) (12Lin D.H. Correia A.R. Cai S.W. Huber F.M. Jette C.A. Hoelz A. Structural and functional analysis of mRNA export regulation by the nuclear pore complex.Nat. Commun. 2018; 9 (29899397)2319 10.1038/s41467-018-04459-3Crossref PubMed Scopus (24) Google Scholar, 13Adams R.L. Mason A.C. Glass L. Aditi Wente S.R. Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.Traffic. 2017; 18 (28869701): 776-79010.1111/tra.12526Crossref PubMed Scopus (29) Google Scholar, 14Kendirgi F. Rexer D.J. Alcázar-Román A.R. Onishko H.M. Wente S.R. Interaction between the shuttling mRNA export factor Gle1 and the nucleoporin hCG1: a conserved mechanism in the export of Hsp70 mRNA.Mol. Biol. Cell. 2005; 16 (16000379): 4304-431510.1091/mbc.e04-11-0998Crossref PubMed Scopus (57) Google Scholar, 15Saavedra C.A. Hammell C.M. Heath C.V. Cole C.N. Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p.Genes Dev. 1997; 11 (9353254): 2845-285610.1101/gad.11.21.2845Crossref PubMed Scopus (111) Google Scholar). Through the interaction with Nup42 and other NPC-associated factors, Gle1B shows steady-state localization to the nuclear rim, whereas Gle1A is predominantly cytoplasmic and only at the nuclear rim when Gle1B is absent (3Aditi Folkmann A.W. Wente S.R. Cytoplasmic hGle1A regulates stress granules by modulation of translation.Mol. Biol. Cell. 2015; 26 (25694449): 1476-149010.1091/mbc.E14-11-1523Crossref PubMed Scopus (25) Google Scholar, 11Kendirgi F. Barry D.M. Griffis E.R. Powers M.A. Wente S.R. An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.J. Cell Biol. 2003; 160 (12668658): 1029-104010.1083/jcb.200211081Crossref PubMed Scopus (46) Google Scholar). Through differing expression levels, protein interactions, and subcellular localization, Gle1A and Gle1B execute unique functions. Gle1B facilitates the terminal step of mRNA export through the NPC by activating DDX19B in an inositol hexakisphosphate (IP6)–dependent manner (4Alcázar-Román A.R. Tran E.J. Guo S. Wente S.R. Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.Nat. Cell Biol. 2006; 8 (16783363): 711-71610.1038/ncb1427Crossref PubMed Scopus (207) Google Scholar, 9Montpetit B. Thomsen N.D. Helmke K.J. Seeliger M.A. Berger J.M. Weis K. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP 6 in mRNA export.Nature. 2011; 472 (21441902): 238-24210.1038/nature09862Crossref PubMed Scopus (161) Google Scholar, 10Weirich C.S. Erzberger J.P. Flick J.S. Berger J.M. Thorner J. Weis K. Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.Nat. Cell Biol. 2006; 8 (16783364): 668-67610.1038/ncb1424Crossref PubMed Scopus (194) Google Scholar, 13Adams R.L. Mason A.C. Glass L. Aditi Wente S.R. Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.Traffic. 2017; 18 (28869701): 776-79010.1111/tra.12526Crossref PubMed Scopus (29) Google Scholar). In the cytoplasm, Gle1A is required for proper stress granule (SG) formation and translation initiation during cellular stress through IP6-independent modulation of DDX3 (3Aditi Folkmann A.W. Wente S.R. Cytoplasmic hGle1A regulates stress granules by modulation of translation.Mol. Biol. Cell. 2015; 26 (25694449): 1476-149010.1091/mbc.E14-11-1523Crossref PubMed Scopus (25) Google Scholar, 5Bolger T.A. Wente S.R. Gle1 is a multifunctional DEAD-box protein regulator that modulates Ded1 in translation initiation.J. Biol. Chem. 2011; 286 (21949122): 39750-3975910.1074/jbc.M111.299321Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 6Bolger T.A. Folkmann A.W. Tran E.J. Wente S.R. The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation.Cell. 2008; 134 (18724935): 624-63310.1016/j.cell.2008.06.027Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 16Aditi Mason A.C. Sharma M. Dawson T.R. Wente S.R. MAPK- and glycogen synthase kinase 3–mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.J. Biol. Chem. 2019; 294 (30429220): 559-57510.1074/jbc.RA118.005749Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In S. cerevisiae, IP6-dependent Gle1 regulation of Dbp5, the ortholog of DDX19B, also plays a role during translation termination (6Bolger T.A. Folkmann A.W. Tran E.J. Wente S.R. The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation.Cell. 2008; 134 (18724935): 624-63310.1016/j.cell.2008.06.027Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). However, a translation termination function for human Gle1A or Gle1B has not been reported. Substantial Gle1 structural and functional analysis maps the residues responsible for Dbp5/DDX19B and IP6 interactions to the C-terminal region, with no contribution of its N-terminal domains to ATPase stimulation (4Alcázar-Román A.R. Tran E.J. Guo S. Wente S.R. Inositol hexakisphosphate and Gle1 activate the DEAD-box protein Dbp5 for nuclear mRNA export.Nat. Cell Biol. 2006; 8 (16783363): 711-71610.1038/ncb1427Crossref PubMed Scopus (207) Google Scholar, 7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 9Montpetit B. Thomsen N.D. Helmke K.J. Seeliger M.A. Berger J.M. Weis K. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP 6 in mRNA export.Nature. 2011; 472 (21441902): 238-24210.1038/nature09862Crossref PubMed Scopus (161) Google Scholar, 12Lin D.H. Correia A.R. Cai S.W. Huber F.M. Jette C.A. Hoelz A. Structural and functional analysis of mRNA export regulation by the nuclear pore complex.Nat. Commun. 2018; 9 (29899397)2319 10.1038/s41467-018-04459-3Crossref PubMed Scopus (24) Google Scholar). Likewise, activation of DDX3 ATPase activity in vitro also requires only the Gle1 C-terminal domain (16Aditi Mason A.C. Sharma M. Dawson T.R. Wente S.R. MAPK- and glycogen synthase kinase 3–mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.J. Biol. Chem. 2019; 294 (30429220): 559-57510.1074/jbc.RA118.005749Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Dbps/DDXs and their specific cofactors are also dynamically regulated by their oligomerization state, which directly alters their intrinsic activity and also provides new binding surfaces for other protein interactions. DDX3 and its S. cerevisiae ortholog Ded1 function as a trimer to unwind RNA duplexes (17Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49 (25039764): 343-36010.3109/10409238.2014.931339Crossref PubMed Scopus (77) Google Scholar, 18Putnam A.A. Gao Z. Liu F. Jia H. Yang Q. Jankowsky E. Division of labor in an oligomer of the DEAD-box RNA helicase Ded1p.Mol. Cell. 2015; 59 (26212457): 541-55210.1016/j.molcel.2015.06.030Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Ded1 oligomerization is disrupted by eIF4G, and the Ded1–eIF4G complex also acts as a functional helicase but with reduced activity as compared with the trimeric Ded1 (18Putnam A.A. Gao Z. Liu F. Jia H. Yang Q. Jankowsky E. Division of labor in an oligomer of the DEAD-box RNA helicase Ded1p.Mol. Cell. 2015; 59 (26212457): 541-55210.1016/j.molcel.2015.06.030Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). DDX3 interaction with Ezrin inhibits its helicase activity and increases its ATPase activity (19Çelik H. Sajwan K.P. Selvanathan S.P. Marsh B.J. Pai A.V. Kont Y.S. Han J. Minas T.Z. Rahim S. Erkizan H.V. Toretsky J.A. Üren A. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level.Mol. Cell Biol. 2015; 35 (26149384): 3145-316210.1128/MCB.00332-15PubMed Google Scholar), with Ezrin's oligomeric state being influenced by phosphorylation (20Poullet P. Gautreau A. Kadaré G. Girault J.A. Louvard D. Arpin M. Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.J. Biol. Chem. 2001; 276 (11468295): 37686-3769110.1074/jbc.M106175200Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). As further examples across a range of species, the cold shock RNA helicase, CsdA, from Escherichia coli requires its C-terminal dimerization domain for activity and stability (21Xu L. Wang L. Peng J. Li F. Wu L. Zhang B. Lv M. Zhang J. Gong Q. Zhang R. Zuo X. Zhang Z. Wu J. Tang Y. Shi Y. Insights into the structure of dimeric RNA helicase CsdA and indispensable role of its C-terminal regions.Structure. 2017; 25 (29107486): 1795-1808.e510.1016/j.str.2017.09.013Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In Thermus thermophilus, HerA exists as a heptameric and a hexameric ring with binding of NurA and nucleotide promoting a hexameric oligomerization state. Additionally, NurA functions as a dimer that tightly binds to hexametric HerA and enhances the affinity of HerA–NurA for dsDNA (22Ahdash Z. Lau A.M. Byrne R.T. Lammens K. Stüetzer A. Urlaub H. Booth P.J. Reading E. Hopfner K.-P. Politis A. Mechanistic insight into the assembly of the HerA–NurA helicase–nuclease DNA end resection complex.Nucleic Acids Res. 2017; 45 (29149348): 12025-1203810.1093/nar/gkx890Crossref PubMed Scopus (10) Google Scholar, 23Klostermeier D. Rudolph M.G. A novel dimerization motif in the C-terminal domain of the Thermus thermophilus DEAD box helicase Hera confers substantial flexibility.Nucleic Acids Res. 2009; 37 (19050012): 421-43010.1093/nar/gkn947Crossref PubMed Scopus (40) Google Scholar). Although the C-terminal region of Gle1 is necessary and sufficient for its modulation of DDX activities, the Gle1 N-terminal region self-associates both in vitro and in vivo and is required for proper Gle1 localization and function (7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Indeed, the Gle1 N-terminal region has multiple reported protein–protein interaction attributes, including a short 29-residue Nup155-binding motif (24Rayala H.J. Kendirgi F. Barry D.M. Majerus P.W. Wente S.R. The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155.Mol. Cell. Proteomics. 2004; 3 (14645504): 145-15510.1074/mcp.M300106-MCP200Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), followed by a 123-residue intrinsically disordered region (IDR) of unknown function and structure (16Aditi Mason A.C. Sharma M. Dawson T.R. Wente S.R. MAPK- and glycogen synthase kinase 3–mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.J. Biol. Chem. 2019; 294 (30429220): 559-57510.1074/jbc.RA118.005749Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) and a 208-residue predicted coiled-coil domain (25Watkins J.L. Murphy R. Emtage J.L.T. Wente S.R. The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export.Proc. Natl. Acad. Sci. U.S.A. 1998; 95 (9618489): 6779-678410.1073/pnas.95.12.6779Crossref PubMed Scopus (72) Google Scholar). Biochemically, large 15–60-nm discs form in vitro from purified recombinant Gle11–360, which are perturbed by a gle1-Finmajor mutation that introduces a proline–phenylalanine–glutamine (PFQ) insertion into the predicted coiled-coil domain (7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In human cells, Gle1 dimerization is observable by FRET and requires the N-terminal region. Deletion of the N-terminal region abolishes Gle1 nuclear rim localization, and correspondingly, nucleocytoplasmic shuttling, mRNA export, and in vitro self-association are all perturbed by the gle1-Finmajor mutation. Demonstrating the critical nature of Gle1 oligomerization for proper mRNA export, the gle1-Finmajor mutation underlies the fatal neurodevelopmental disease lethal congenital contracture syndrome-1 (26Nousiainen H.O. Kestilä M. Pakkasjärvi N. Honkala H. Kuure S. Tallila J. Vuopala K. Ignatius J. Herva R. Peltonen L. Mutations in mRNA export mediator GLE1 result in a fetal motoneuron disease.Nat. Genet. 2008; 40 (18204449): 155-15710.1038/ng.2007.65Crossref PubMed Scopus (133) Google Scholar). Although studies in S. cerevisiae suggest that Gle1's roles in translation initiation and termination are not dependent on the coiled-coil domain, this has not been investigated in human cells. Moreover, human Gle1's N-terminal region contains a much longer IDR and is the site of dynamic, stress-induced phosphorylation that alters the formation of Gle1 oligomers in vitro (16Aditi Mason A.C. Sharma M. Dawson T.R. Wente S.R. MAPK- and glycogen synthase kinase 3–mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.J. Biol. Chem. 2019; 294 (30429220): 559-57510.1074/jbc.RA118.005749Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), suggesting that other regions of the N terminus contribute to oligomerization. Overall, further studies are needed to obtain a detailed understanding of the properties governing Gle1 self-association and its effect on isoform-specific functions. Here we investigate the biochemical properties of Gle1 oligomerization and test how disrupting proper Gle1 oligomerization influences Gle1 function in many aspects of RNA metabolism. We find that two distinct N-terminal regions contribute to the overall oligomerization state of Gle1. The coiled-coil domain self-associates in a parallel orientation but is not sufficient to generate high-molecular-weight oligomers. Conversely, an aggregation-prone region in the unstructured IDR is necessary for high-molecular-weight oligomerization. Mutations engineered to result in specific amino acid changes disrupt these two oligomerization domains and distinct functions in vivo. We show that proper Gle1 oligomerization is essential for proper Gle1 subcellular localization and function in mRNA export and heat-shock stress response. Our prior studies show that perturbation of the predicted N-terminal coiled-coil domain by the lethal congenital contracture syndrome 1–linked PFQ insertion impedes gle1-Finmajor oligomerization and nucleocytoplasmic shuttling, as well as poly(A)+ RNA export (7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The N-terminal Gle1 region also contains a Nup155-binding domain, a predicted IDR, and a recently reported stress-induced phosphorylation cluster (Fig. 1A) (16Aditi Mason A.C. Sharma M. Dawson T.R. Wente S.R. MAPK- and glycogen synthase kinase 3–mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.J. Biol. Chem. 2019; 294 (30429220): 559-57510.1074/jbc.RA118.005749Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 24Rayala H.J. Kendirgi F. Barry D.M. Majerus P.W. Wente S.R. The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155.Mol. Cell. Proteomics. 2004; 3 (14645504): 145-15510.1074/mcp.M300106-MCP200Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To investigate the mechanism of human Gle1 self-association, we employed a set of biochemical approaches. First, using MARCOIL (27Delorenzi M. Speed T. An HMM model for coiled-coil domains and a comparison with PSSM-based predictions.Bioinformatics. 2002; 18 (12016059): 617-62510.1093/bioinformatics/18.4.617Crossref PubMed Scopus (328) Google Scholar) to more accurately delineate the boundaries of the predicted coiled-coil domain, we found a near 100% probability of coiled-coil formation spanning amino acids 152–360 in the Gle1 N-terminal region (Fig. 1B). To date, no high-resolution structural studies are reported for the N-terminal Gle1 regions. Coiled-coil domains associate in either a parallel or anti-parallel orientation (28Burkhard P. Stetefeld J. Strelkov S.V. Coiled coils: a highly versatile protein folding motif.Trends Cell Biol. 2001; 11 (11166216): 82-8810.1016/S0962-8924(00)01898-5Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). To roughly discern how Gle1 domains are relatively positioned in a Gle1 oligomer, we probed the orientation required for Gle1 self-association by utilizing a technique termed Assessment tool for homodimeric Coiled-Coil ORientation Decision (ACCORD) (29Kim B.-W. Jung Y.O. Kim M.K. Kwon D.H. Park S.H. Kim J.H. Kuk Y.-B. Oh S.-J. Kim L. Kim B.H. Yang W.S. Song H.K. ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils.Sci. Rep. 2017; 7 (28266564)43318 10.1038/srep43318Crossref PubMed Scopus (5) Google Scholar). This method is based on forced dimerization by the E. coli protein SspB when the respective coiled-coil domain being analyzed is expressed as a fusion protein with SspB. The resulting purified chimeric protein is then analyzed by size-exclusion chromatography multiangle light scattering (SEC-MALS). Coiled-coil domains associating in a parallel orientation result in a single dimerization event, whereas anti-parallel orientation results in two dimerization interfaces that produce a tetramer or higher-ordered complex. Analysis of the purified SspB-Gle1152–360 fusion protein by SEC-MALS revealed a sharp peak with a calculated molecular weight in agreement with a dimer (Fig. 1C). Therefore, we concluded that the Gle1 coiled-coil domain self-associates in a parallel orientation. Coiled-coil domains are also predicted by a common heptad repeat of (a-b-c-d-e-f-g)n, wherein the a and d positions are predominantly hydrophobic residues that form the oligomer's hydrophobic core (28Burkhard P. Stetefeld J. Strelkov S.V. Coiled coils: a highly versatile protein folding motif.Trends Cell Biol. 2001; 11 (11166216): 82-8810.1016/S0962-8924(00)01898-5Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). To identify the heptad repeats present in the Gle1 coiled-coil domain, the amino acid sequence from 1 to 360 was submitted to the MARCOIL web-based server for comparison with a database of known parallel-oriented coiled-coil interactions (27Delorenzi M. Speed T. An HMM model for coiled-coil domains and a comparison with PSSM-based predictions.Bioinformatics. 2002; 18 (12016059): 617-62510.1093/bioinformatics/18.4.617Crossref PubMed Scopus (328) Google Scholar). MARCOIL confirmed the presence of the canonical heptad repeat in Gle1 (Fig. 1D). The second span of heptad repeats contain a two-residue insertion (a-b-c-d-c-d-e-f-g) and a two-residue deletion (a-b-c-f-g), which could alter structural characteristics of the coiled-coil domain (30Hartmann M.D. Mendler C.T. Bassler J. Karamichali I. Ridderbusch O. Lupas A.N. Hernandez Alvarex B. α/β coiled coils.eLife. 2016; 5 (26771248)e11861 10.7554/eLife.11861Crossref PubMed Scopus (2) Google Scholar). Based on MARCOIL predicted amino acids in the hydrophobic core, we mutated the coding sequence for a series of hydrophobic residues in the a and d positions in the context of Gle11–360 to result in the introduction of aspartic acid residues. The resulting purified proteins were analyzed by SDS-PAGE and native PAGE (Fig. 1, E and F, respectively). As shown by native PAGE, successive introduction of charged amino acids into the predicted hydrophobic core resulted in faster electrophoretic migration, with the 8D protein fully collapsing the slower migrating bands that are indicative of Gle1 oligomers. Thus, Gle1 oligomerization in vitro required the interaction of parallel-oriented coiled-coil domains. Analysis of recombinant Gle11–360 by SEC and negative stain EM shows it elutes in the void volume as large, higher-ordered oligomeric discs of 15–60-nm diameter (7Folkmann A.W. Collier S.E. Zhan X. Aditi Ohi M.D. Wente S.R. Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.Cell. 2013; 155 (24243016): 582-59310.1016/j.cell.2013.09.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To test whether the coiled-coil domain alone is sufficient to form these large discs, we compared the SEC profiles of Gle1152–360 and Gle11–360. As shown in Fig. 1G, Gle11–360 eluted in the void volume, consistent with previous reports. However, Gle1152–360 did not elute in the void volume but rather eluted as a sharp peak in later fractions (Fig. 1G). This suggested that the coiled-coil domain alone is not sufficient to form high-molecular-weight oligomers. Because expression of Gle1152–360, containing only the coiled-coil domain, did not produce high-molecular-weight oligomers, we speculated that a different region in the N-terminal region of Gle1 mediates the disc formation. The IDR in the N-terminal region was analyzed for molecular recognition features (DICHOT) (31Fukuchi S. Hosoda K. Homma K. Gojobori T. Nishikawa K. Binary classification of protein molecules into intrinsically disordered and ordered segments.BMC Struct. Biol. 2011; 11 (21693062): 2910.1186/1472-6807-11-29Crossref PubMed Scopus (50) Google Scholar), short linear peptide motifs (ELM Prediction) (32Gouw M. Michael S. Sámano-Sánchez H. Kumar M. Zeke A. Lang B. Bely B. Chemes L.B. Davey N.E. Deng Z. Diella F. Gürth C.-M. Huber A.-K. Kleinsorg S. Schlegel L.S. et al.The eukaryotic linear motif resource: 2018 update.Nucleic Acids Res. 2018; 46 (29136216): D428-D43410.1093/nar/gkx1077Crossref PubMed Scopus (104) Google Scholar), prion-like domains (PrionW) (33Zambrano R. Conchillo-Sole O. Iglesias V. Illa R. Rousseau F. Schymkowitz J. Sabate R. Daura X. Ventura S. PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores.Nucleic Acids Res. 2015; 43 (25977297): W331-